Immediate protection against pathogens via mva

ABSTRACT

The invention relates to methods and kits comprising poxviruses including, but not limited to modified vaccinia virus Ankara (MVA) and uses thereof to provide immediate protection against pathogens. Poxviruses including, but not limited to MVA can be delivered to a host animal just prior to or after exposure to a pathogen and provide protection against the pathogen.

FIELD OF THE INVENTION

The invention relates to the use of modified vaccinia virus Ankara (MVA)to provide immediate protection against pathogens.

BACKGROUND OF THE INVENTION

Poxviruses, including the causative agent of smallpox, Variola virus(VARV), are highly pathogenic double stranded (ds) DNA viruses. It isestimated that smallpox has caused more than 300 million deaths in the20th century alone. Even though traditional vaccination programs haveeradicated VARV as a natural pathogen, it remains that enhancing theknowledge of mechanisms of its infections and/or protection may beessential given the scenarios of zoonotic poxvirus infections (e.g.monkeypox), the re-emergence of VARV by accidental release, or thepossibility of terrorist attacks with poxviruses.

The genus Orthopoxvirus contains several related viruses based ongenetic similarity and immunological cross-reactivity, including VARV,the causative agent of human smallpox, ectromelia virus (ECTV) causingmousepox, cowpox virus (CPXV), monkeypox virus (MPXV), camelpox virus(CMPV), and vaccinia virus (VACV). Among these ECTV, MPXV, and VACV areused in animals as model infections for human smallpox. A number of VACVstrains have been used in mice and large amounts of evidence on theimmune responses induced and on the immune suppressing mechanismsemployed by poxviruses have been elucidated with VACV. However, VACV isnot a natural pathogen of mice and high doses are needed to lethallyinfect mice, even though mouse-adapted strains like VACV Western Reserve(WR) are commonly used (Williamson et al., J. Gen. Virol. 71:2761-2767(1990)). MPXV in monkeys has the advantage that monkeys areevolutionarily much closer to humans. However, as with the VACV model inmice, non-physiological high viral doses are needed to lethally infectmonkeys. Therefore, both animal models are regarded to reflect more thelate stage of a VARV infection in humans (Fenner, F., Henderson, D. A.,Arita, I., Jezek, Z., & Ladnyi, I. D. Smallpox and its eradication.Geneva: World Health Organization (1988); Mortimer, P. P. Clin. Infect.Dis. 36, 622-629 (2003)). Among the orthopoxvirus infection models, ECTVinfection of mice stands out because it is a species-specific virusinfecting its natural host and can cause fatal outcomes afterinoculation with low virus doses, features that have also been describedin VARV infection of humans (Fenner et al., 1988; Esteban, D. J., andBuller, R. M., J. Gen. Virol. 86:2645-2659 (2005)). For these reasons,this model is the closest model to human infection by VARV. Pathogensare detected by the immune system via pattern recognition receptors(PRR). Among the latter is the family of Toll-like receptors (TLR).TLR7, and TLR8 and 9 recognize the nucleic acids RNA and DNA,respectively (Hemmi, H. et al., Nature 408, 740-745 (2000); Diebold, S.S. et al. Science 303, 1529-1531 (2004); Heil, F. Science 303, 1526-1529(2004)). Double stranded DNA (dsDNA) viruses, like herpesviruses oradenoviruses, can be detected via TLR9-dependent pathways(Basner-Tschakarjan, E. et al., J. Gene Med. 8, 1300-1306 (2006); Lund,J. et al., J. Exp. Med. 198, 513-520 (2003); Krug, A. et al. Blood 103,1433-1437 (2004); Hochrein, H. et al. Proc. Natl. Acad. Sci. U.S.A. 101,11416-11421 (2004); Tabeta, K. et al. Proc. Natl. Acad. Sci. U.S.A. 101,3516-3521 (2004)). However, potent alternative recognition pathwaysexist, possibly explaining why previous viral infection studies havedemonstrated no or only mild increases of susceptibility in the absenceof TLR9 (Hochrein, H. et al. Proc. Natl. Acad. Sci. U.S.A. 101,11416-11421 (2004); Krug, A et al. Blood 103, 1433-1437 (2004); Zhu, J.et al. Blood 109, 619-625 (2007); Delale, T. J. Immunol. 175, 6723-6732(2005); Tabeta, K. et al. Proc. Natl. Acad. Sci. U.S.A. 101, 3516-3521(2004)).

Whereas many TLR are located at the outer membrane of the cell tomonitor the extracellular space for danger signals like bacterial cellwall components, a group of TLR consisting of TLR 3, 7, 8, and 9 areassociated with the endosome and monitor the endosomal lumen for nucleicacids (Wagner, H., and Bauer, S., J. Exp. Med. 203:265-268 (2006)). TheTLR 3, 7, and 8 recognize RNA, whereas TLR9 recognizes DNA (Diebold etal., Science 303: 1529-153 (2004); Heil et al., Science 303:1526-1529(2004); Hemmi et al., Nature 408:740-745 (2000); Alexopoulou et al.,Nature 413:732-738 (2001)).

Expression of TLR9 differs within species. Whereas in humans B-cells andplasmacytoid DC (pDC), but not conventional DC (cDC), express andrespond to TLR9 stimulation, TLR9 expression in mice is less restricted.Besides B-cells and pDC, mouse cDC and even macrophages are known toexpress TLR9 and respond to TLR9 ligation (Hochrein et al., Hum.Immunol. 63:1103-1110 (2002)). The natural ligand for TLR9 wasoriginally defined to be genomic bacterial DNA, whereas oligonucleotidescontaining unmethylated CpG motifs adjoined by species specific motifsand often phosphorothioate-stabilized (CpG-ODN), were established asartificial ligands for TLR9 (Hemmi et al., 2000; Bauer et al., Proc.Natl. Acad. Sci. U.S.A 98:9237-9242 (2001)).

Meanwhile, the list of CpG containing natural and artificial ligands hasincreased to bacterial plasmid DNA and several types of CpG-ODN withdifferences in their chemical composition, as well as drasticdifferences in biological effects including IFN-I inducing capacity(Spies et al., J. Immunol. 171:5908-5912 (2003); Krieg, A. M. Nat. Rev.Drug Discov. 5:471-484 (2006)). Under conditions of enhanced uptake, nonCpG-containing or fully methylated DNA as well as vertebrate DNA havealso been shown to act as TLR9 agonists (Yasuda et al., J. Immunol.174:6129-6136 (2005); Means et al., J. Clin. Invest 115:407-417 (2005)).

Poxviruses have evolved multiple strategies for immune suppression,substantiated by the fact that poxvirus genomes encode numerousmolecules with immunosuppressive function. Among these are solublecytokine and chemokine receptors and a multitude of molecules thatinterfere with intracellular signaling cascades (Seet, B. T. et al.Annu. Rev. Immunol. 21, 377-423 (2003)). Recently, molecules expressedby poxviruses have been shown to target members of the TLR signalingcascade, suggesting a role for TLR-dependent recognition pathways forpoxviruses (Bowie, A. et al. Proc. Natl. Acad. Sci. U.S.A. 97,10162-10167 (2000)). In fact, a role for TLR2 in the recognition ofvaccinia viruses (VACV) was proposed (Zhu, J. et al. Blood 109, 619-625(2007)).

Modified Vaccinia Ankara (MVA) virus is related to Vaccinia virus, amember of the genera Orthopoxvirus in the family of Poxviridae. MVA hasbeen generated by 516 serial passages on chicken embryo fibroblasts ofthe Ankara strain of vaccinia virus (CVA) (for review see Mayr, A., etal. Infection 3, 6-14 (1975)). As a consequence of these long-termpassages, the resulting MVA virus deleted about 31 kilobases of itsgenomic sequence and, therefore, was described as highly host cellrestricted to avian cells (Meyer, H. et al., J. Gen. Virol. 72,1031-1038 (1991)). It was shown, in a variety of animal models, that theresulting MVA was significantly avirulent (Mayr, A. & Danner, K. Dev.Biol. Stand. 41: 225-34 (1978)). Additionally, this MVA strain has beentested in clinical trials as vaccine to immunize against the humansmallpox disease (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167,375-390 (1987), Stickl et al., Dtsch. Med. Wschr. 99, 2386-2392 (1974)).These studies involved over 120,000 humans, including high riskpatients, and proved that, compared to Vaccinia based vaccines, MVA haddiminished virulence or infectiousness while it maintained goodimmunogenicity. In the decades that followed, MVA has been engineered touse it as viral vector for recombinant gene expression and as arecombinant vaccine (Sutter, G. et al., Vaccine 12: 1032-40 (1994)).

In this respect, it is most astonishing that, even though Mayr et al.demonstrated during the 1970s that MVA is highly attenuated andavirulent in humans and mammals, some recently reported observations(Blanchard et al. J. Gen. ViroL 79, 1159-1167 (1998); Carroll & Moss,Virology 238, 198-211 (1997); U.S. Pat. No. 5,185,146; Ambrosini et al.,J. Neurosci. Res. 55(5), 569 (1999)) have shown that MVA is not fullyattenuated in mammalian and human cell lines since residual replicationmight occur in these cells. It is assumed that the results reported inthese publications have been obtained with various strains of MVA, sincethe viruses used essentially differ in their properties, particularly intheir growth behavior in various cell lines.

Growth behavior is recognized as one of several indicators for virusattenuation. Generally, a virus strain is regarded as attenuated if ithas lost its capacity or only has reduced capacity to reproductivelyreplicate in host cells. The above-mentioned observation, that MVA isnot completely replication incompetent in human and mammalian cells,brings into question the absolute safety of MVA as a human vaccine or avector for recombinant vaccines.

Particularly, for a vaccine as well as for a recombinant vaccine, thebalance between the efficacy and the safety of the vaccine vector virusis extremely important.

As described in WO publication 02/42480, novel MVA strains with enhancedsafety have been developed. These strains are characterized by having atleast one of the following advantageous properties:

-   -   capability of reproductive replication in vitro in chicken        embryo fibroblasts (CEF), but no capability of reproductive        replication in a human cell line, as in the human keratinocyte        cell line HaCaT, the human embryo kidney cell line 293, the        human bone osteosarcoma cell line 143B, and the human cervix        adenocarcinoma cell line HeLa;    -   (ii) failure to replicate in a mouse model that is incapable of        producing mature B and T cells and as such is severely immune        compromised and highly susceptible to a replicating virus; and    -   (iii) induction of at least the same level of specific immune        response in vaccinia virus prime/vaccinia virus boost regimes        when compared to DNA-prime/vaccinia virus boost regimes.

One of the developed strains has been deposited at the EuropeanCollection of Animal Cell Cultures (ECACC) with the deposit numberV00083008. This strain is referred to as “MVA-BN” throughout thespecification of WO 02/42480.

The terms “not capable of reproductive replication” or “replicationincompetent” mean that the virus shows an amplification ratio of lessthan 1 in human cell lines, such as the cell lines 293 (ECACC No.85120602), 143B (ECACC No. 91112502), HeLa (ATCC No. CCL-2) and HaCat(Boukamp et al., J. Cell Biol. 106(3): 761-71 (1988)), under theconditions as outlined in Example 1 of WO 02/42480 for some specific MVAstrains.

According to WO 02/42480, “failure to replicate in vivo” refers toviruses that do not replicate in humans and in the mice model asdescribed in the WO 02/42480 publication.

There have been numerous reports suggesting that prime/boost regimesusing MVA as a delivery vector induce poor immune responses and areinferior to DNA-prime/MVA-boost regimes (Schneider et al., Nat. Med. 4;397-402 (1998)). In all these studies, MVA strains have been used thatare different from the vaccinia viruses as developed according to WO02/42480. As an explanation for the poor immune response obtained whenMVA was used for prime and boost administration, it has beenhypothesized that antibodies generated to MVA during theprime-administration neutralize the MVA given in the secondimmunization, preventing an effective boost of the immune response. Incontrast, DNA-prime/MVA-boost reaimes are reported to be superior atgenerating high avidity responses, because this regime combines theability of DNA to effectively prime the immune response with theproperties of MVA to boost this response in the absence of apre-existing immunity to MVA. Clearly, if a pre-existing immunity to MVAand/or vaccinia prevents boosting of the immune response, then the useof MVA as a vaccine or therapeutic would have limited efficacy,particularly in the individuals that have been vaccinated againstsmallpox. However, the vaccinia virus strains according to WO 02/42480,as well as corresponding recombinant viruses harbouring heterologoussequences, can be used to efficiently first prime and then boost immuneresponses in native animals as well as in animals with a pre-existingimmunity to poxviruses. Thus, the developed strains as described in WO02/42480 induce at least substantially the same level of immunity invaccinia virus prime/vaccinia virus boost regimes compared toDNA-prime/vaccinia virus boost regimes.

A vaccinia virus is regarded as inducing at least substantially the samelevel of immunity in vaccinia virus prime/vaccinia virus boost regimeswhen compared to DNA-prime/vaccinia virus boost regimes if the CTLresponse as measured in one of the two, or even in both assays, asdescribed in WO 02/42480 is at least substantially the same in vacciniavirus prime/vaccinia virus boost regimes when compared toDNA-prime/vaccinia virus boost regimes.

The growth behavior of the vaccinia viruses developed according to WO02/42480, in particular the growth behavior of MVA-BN®, indicates thatthe strains are far superior to any other so far characterized MVAisolate regarding attenuation in human cell lines and failure of in vivoreplication. The strains are therefore ideal candidates for thedevelopment of safer products such as vaccines or pharmaceuticals.

An immune response is raised by the immune system when a foreignsubstance or microorganism enters the organism. By definition, theimmune response is divided into a specific and an unspecific reaction,although both are closely cross linked. The unspecific immune responseis the immediate defence against a wide variety of foreign substancesand infectious agents. The specific immune response is the defenceraised after a lag phase, when the organism is challenged with asubstance for the first time. The specific immune response is highlyefficient, and is responsible for the fact that an individual whorecovers from a specific infection is protected against this specificinfection. Thus, a second infection with the same or a very similarinfectious agent causes much milder symptoms or no symptoms at all,since there is already a “pre-existing immunity” to this agent. Suchimmunity and the immunological memory persist for a long time, in somecases even lifelong. Accordingly, the induction of an immunologicalmemory can result from vaccination.

The “immune system” means a complex organ involved in the defence of theorganism against foreign substances and micro-organisms. The immunesystem comprises a cellular part comprising several cell types, such as,e.g., lymphocytes and other cells derived from white blood cells, and ahumoral part comprising small peptides and complement factors.

Traditional vaccination strategies are able to induce effective and longlasting protection by inducing adaptive immune responses (antibodies,CTL). However, substantial protection can only be achieved after severaldays to months, optimally with a boost regime, which leaves theindividual susceptible to infection during that time.

MVA is a non-replicating virus in humans, which can be administered topeople with various degrees of immune deviation (HIV, allergies, atopicdermatitis, certain drug treatments), even via systemic applicationroutes. In these cases of immune deviation, a specialized anti-viralimmune cell population (pDC) is reduced in number or affected in itsfunctional properties, which may increase the risk for viral infection.

The current view of protection against deadly poxviruses is viavaccinations. For these approaches, individuals are exposed to anattenuated (less pathogenic) poxvirus before the potential exposure to apathogenic poxvirus. Vaccination induces adaptive immune responses likeKiller T cells (CTL) and antibodies and a memory against the relatedvaccinating virus. This results in some reactivity against thepathogenic virus, leading to protection and quick resurrection of thememory responses upon repeated exposure. However, adaptive immuneresponses need time to develop, and are optimal after boosting theimmune response with repetitive application of the vaccinating virus.

Recently, it was reported that, employing MVA as vaccinating virusseveral days (at latest 2 days) before exposure with the Vaccinia virusWestern Reserve strain (VV-WR), some protection can be achieved(WO2006/089690, which is hereby incorporated by reference in itsentirety). Similar results have been published by another group, whichfurther demonstrated post-exposure treatment failed to protect animals.(Staib, C. et al. J. Gen. Virol 87, 2917-2921 (2006)). The protectionlevels were 1×LD50 if vaccinated 2 days before exposure to VV-WR and12.5×LD50 if vaccinated 3 days before exposure to VV-WR. (Id.)

Stittelaar et al., Nature 439:745-748 (2006) compared the effects ofantiviral treatment and smallpox vaccination upon lethal monkeypox virusinfection. They reported that when monkeys were vaccinated 24 h aftermonkeypox virus infection, using a standard human dose of a currentlyrecommended smallpox vaccine (Elstree-RIVM), no significant reduction inmortality was observed.

Thus, there is a need in the art for reagents and methods for immediateprotection against pathogens, such as smallpox.

BRIEF SUMMARY OF THE INVENTION

The invention encompasses a method for inducing an immune responseagainst an infectious agent in an animal, comprising administering tothe animal an immunogenic composition comprising a poxvirus between 36hours prior to infection with the infectious agent and 72 hours afterinfection with the infectious agent.

Furthermore, the invention encompasses the use of a poxvirus for thepreparation of an immunogenic composition comprising said poxvirus forinducing an immune response against an infectious agent in an animal,wherein said immunogenic composition is to be administered to the animalbetween 36 hours prior to infection with the infectious agent and 72hours after infection with the infectious agent.

The invention also encompasses a poxvirus for inducing an immuneresponse against an infectious agent in an animal. Furthermore, theinvention encompasses an immunogenic composition comprising saidpoxvirus for inducing an immune response against an infectious agent inan animal. In a preferred embodiment, said poxvirus or immunogeniccomposition comprising said poxvirus is to be administered to the animalbetween 36 hours prior to infection with the infectious agent and 72hours after infection with the infectious agent.

In a preferred embodiment, the poxvirus is administered between 36 hoursprior to infection with the infectious agent and 48 hours afterinfection with the infectious agent.

In a preferred embodiment, the poxvirus is replication incompetent inthe animal.

In a preferred embodiment, the poxvirus is a Modified Vaccinia VirusAnkara (MVA).

In a preferred embodiment, the animal is a human.

In a preferred embodiment, the infectious agent is a replicationcompetent poxvirus.

In one embodiment, the MVA is administered in a dose of 10⁵ to 5×10⁸TCID50. In a preferred embodiment, the MVA is administeredintravenously, intranasally, intramuscularly, or subcutaneously.

In one embodiment, the MVA is MVA-BN®. The MVA can be a recombinant MVAand can comprise at least one heterologous nucleic acid sequence codingfor at least one antigenic epitope. The antigenic epitope can be anantigenic epitope of the infectious agent. The infectious agent can beselected from viruses, fungi, pathogenic unicellular eukaryotic andprokaryotic organisms, and parasitic organisms. In a preferredembodiment, the virus is selected from Influenza virus, Flavivirus,Paramyxovirus, Hepatitis virus, human immunodeficiency virus, andviruses causing hemorrhagic fever. In another preferred embodiment, theinfectious agent is bacillus anthracis.

In one embodiment, the immunogenic composition is administered between24 hours prior to infection with the infectious agent and 24 hours afterinfection with the infectious agent. The immunogenic composition can beadministered at the same time as infection with the infectious agent.The administration to the animal of the immunogenic compositioncomprising an MVA can be between 0 and 24 hours prior to infection withan infectious agent or between 0 and 48 hours after infection with aninfectious agent.

The invention also encompasses a kit for inducing an immune responseagainst an infectious agent in an animal, including a human, comprisingan immunogenic composition comprising a poxvirus as active substance,wherein said poxvirus is replication incompetent in said animal,including a human.

Preferably, said kit comprises an immunogenic composition comprising anMVA, and instructions to deliver the immunogenic composition at a timepoint between 0 hours and 36 hours prior to exposure to an infectiousagent or at a time point between 0 hours and 72 hours after exposure toan infectious agent. In one embodiment, the MVA is MVA-BN® at dose of10⁵ to 5×10⁸ TCID50.

In one embodiment, the infectious agent is smallpox. In anotherembodiment, the infectious agent is bacillus anthracis. The infectiousagent may also be comprised within the kit in a separate vial.

In a preferred embodiment, the invention encompasses a kit comprising animmunogenic composition comprising an MVA and instructions to deliverthe immunogenic composition as soon as possible after exposure tosmallpox.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is more completely understood with reference to thedrawings, in which:

FIG. 1 a and b depict analysis of Dendritic Cell (DC) maturation inresponse to active or inactivated poxviruses. Flow cytometry histogramsshowing expression of CD40 or CD69 on FL-DC after incubation with active(left panel) or inactive (right panel) poxviruses (shaded histograms) asindicated. a) CVA, ECTV, CPXV. b) MVA, SFV, CNPV or without stimulation(empty histograms). One representative experiment of at least three(CVA, ECTV, MVA) or two (CPXV, SFV, CNPV) experiments is shown.

FIG. 2 a-e depict response of TLR9-deficient or wild type DC to poxvirusinfection in vitro. a) Flow cytometry histograms showing expression ofCD40 or CD69 on FL-DC of wild type (left panel) or TLR9-deficient mice(right panel) after incubation with CVA, ECTV or MVA as indicated(shaded histograms) or without stimulation (empty histograms). Thesimilar histograms for the wild type activation are part of FIG. 1. b)FL-DC or c) GM-DC of TLR9-deficient (empty column) or wild type mice(filled column) were stimulated with active (left panel) orUV-inactivated (right panel) MVA and the supernatants were analyzed forIFN-α and IL-6 by ELISA. d) Sorted FL-pDC and cDC of TLR9-deficient orwild type mice as indicated were stimulated with MVA (filled column) orECTV (empty column) and supernatants were analyzed for IFN-α and IL-6 byELISA. e) Total bone marrow cells were stimulated with active(diagonally striped column) or UV-inactivated (horizontally stripedcolumn) MVA or CpG-2216 (black column) and the supernatants wereanalyzed for IFN-α by ELISA. Representative experiments of at least twoexperiments are shown.

FIG. 3 a and b depict survival of wild type and TLR9-deficient mice toECTV infection. Wild type mice (a) and TLR9-deficient mice (b) were i.n.infected with varying doses of ECTV (TCID50 per mouse) as indicated andsurvival was monitored for at least 4 weeks. The experiments wereperformed with the numbers of mice as indicated and data represent atleast 3 individual experiments for each viral dose in wild type mice (a)or 7 experiments for the dose of 1E+02 for TLR9-KO mice (b) and oneexperiment for the other doses (b). The data for the dose of 1E+04 inwild type (a) and 1E+02 in TLR9-KO mice (b) include death control miceof other experiments.

FIG. 4 depicts that MVA protects wild type mice if given simultaneouslywith lethal doses of ECTV. Wild type mice were i.n. infected with lethaldoses of ECTV as indicated and simultaneously i.n. inoculated with(black symbols) or without (grey square) 1E+08 TCID50 of MVA andsurvival was monitored for 4 weeks. The experiments were performed withthe numbers of mice as indicated and data represent the results of twoindividual experiments.

FIG. 5 depicts that MVA protects TLR9 deficient mice if givensimultaneously with lethal doses of ECTV. TLR9-deficient mice were i.n.infected with lethal doses of ECTV as indicated and simultaneously i.n.inoculated with (black symbols) or without (grey square) 1E+08 TCID50 ofMVA and survival was monitored for 4 weeks. The experiment was performedwith the numbers of mice as indicated.

FIG. 6 a and b depict that MVA protects TLR9 deficient and wild typemice against lethal ECTV challenge if applied subcutaneously. a) TLR9deficient mice were i.n. infected with 1E+02 TCID50 of ECTV andsimultaneously s.c. inoculated with 1E+08 TCID50 of MVA (black squares)or without (grey square). b) Wild type mice were i.n. infected with1E+04 TCID50 of ECTV and simultaneously s.c. inoculated with 1E+08TCID50 of MVA (black squares) or with the corresponding amount of 1E+08TCID50 of UV-inactivated CVA (black triangle). Survival was monitoredfor 4 weeks. The experiments were performed with the numbers of mice asindicated and data represent the results of two individual experimentsfor wild type mice with MVA and one experiment for wild type mice withUV-inactivated CVA or for TLR9 deficient mice with MVA.

FIG. 7 depicts that MVA partially protects IFN-I-R-deficient mice ifgiven simultaneously with lethal doses of ECTV. IFN-I-R-deficient micewere i.n. infected with lethal doses of ECTV as indicated andsimultaneously i.n. inoculated with 1E+08 TCID50 of MVA (black symbols)or without (grey symbols) and survival was monitored for 4 weeks. Theexperiments were performed with the numbers of mice as indicated anddata represent the results of at least two individual experiments forthe challenge dose of (1E+02 and 1E+03) or one experiment for thechallenge dose of 1E+04 and 1E+05.

FIG. 8 depicts long time survival to ECTV infection even in the presenceof MVA depends on adaptive immune responses. RAG-1 deficient mice werei.n. infected with doses of ECTV as indicated and simultaneously i.n.inoculated with 1E+08 TCID₅₀ of MVA (black symbols) or without (greysymbols) and survival was monitored for 4 weeks. The experiment wasperformed with the numbers of mice as indicated.

FIG. 9 a and b depict that MVA therapeutically protects TLR9-deficientmice if applied after infection with a lethal dose of ECTV.TLR9-deficient mice were i.n. infected with 1E+02 TCID₅₀ ECTV. After theindicated times of 24 hrs (a) or 48 hrs or 72 hrs (b) the ECTV infectedmice were i.n. inoculated with 1E+08 TCID₅₀ of MVA (black symbols) orwithout (grey square) and survival was monitored for 4 weeks. Theexperiments were performed with the numbers of mice as indicated anddata show the cumulated results of 3 individual experiments (a) or oneexperiment (b). Note that the 9 control mice of a) include the 3 controlmice of b).

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the present embodiments(exemplary embodiments) of the invention, examples of which areillustrated in the accompanying drawings and Examples section.

An animal model applying intranasaly the species specific and highlypathogenic mousepoxvirus Ectromelia was used. Like Variola, which ishighly specific for the human species, Ectromelia is highly specific formice. Both Ectromelia and Variola employ a large panel ofimmune-suppressive strategies and, when compared to other pathogenicpoxviruses e.g. “Vaccinia virus Western Reserve”, they areevolutionarily more distant to MVA. Furthermore, both viruses are highlypathogenic (low virus numbers are able to establish infection and causedeath in many of the infected individuals) in their specific hosts.Importantly, both Variola and Ectromelia, are able to infect their hostsvia the respiratory route as a natural way of infection. For thesereasons, Ectromelia infection in mice is a good model system forinfection of humans by Variola.

The experiments in animals demonstrate that the co-administration of MVAtogether with a highly lethal dose of mousepoxvirus protects miceimmediately against the deadly exposure. Immune competent mice survivedthe infection under these conditions when exposed to at least 47 foldthe lethal dose of Ectromelia.

In addition, immune compromised mice were employed in the Ectromeliainfection model. In one model, the mice lack the antiviral receptor TLR9(TLR9-KO). The pDC of the TLR9-KO mice can not respond in the usualanti-viral way to DNA and DNA viruses. It was found that these mice havea drastic increase in susceptibility to Ectromelia infection (more than100-fold more susceptible than immune competent mice).

With research in vitro with isolated cells, it was found that MVA is notas dependent on this specialized anti-viral pDC. The data describedherein showed that MVA can employ additional immune stimulatory pathwaysthat result in the induction of antiviral mechanisms. Thus, it wasanalyzed whether that MVA would also be able to protect TLR9-KO mice viathe alternative pathways that were found in vitro. Indeed, applicationof MVA at the same time as a highly deadly dose of Ectromelia protectedTLR9-KO mice immediately against at least 500-fold the lethal dose ofEctromelia.

Another immune compromised mouse model was tested that lacksresponsiveness to type I interferons (IFN-I). These cytokines arebelieved to be essential for survival of viral infection in general.Unexpectedly, application of MVA at the time of infection with a deadlydose of Ectromelia showed some protection. Thus, this invention protectsnormal as well as several immune compromised individuals againstotherwise deadly pathogens.

In contrast to prior studies, the data described herein with Ectromeliashow that immediate protection can be achieved (giving protecting MVA atthe same time as, or after, the pathogenic poxvirus). The level ofprotection is not only reached much earlier (at the same time/after vs.at the latest 2 days before), but is also much more effective. In immunecompetent mice, the protection factor exceeds 47×LD50. Furthermore, itis demonstrated that the protection in immune compromised mice exceedsthe factor of 500×LD50. Ectromelia infection in mice is the bestinfection model currently available for correlations to Variolainfections in humans.

It is shown here that poxviruses are recognized via TLR9 dependent andindependent recognition pathways. Herein, it is demonstrated thatpathogenic poxviruses like Ectromelia virus effectively suppress therecognition via the TLR9 independent pathway but are still recognized tosome extent via TLR9.

Plasmacytoid DC (pDC) are the only cells which produce large amounts ofthe antiviral and immune regulating cytokines Type I Interferons (IFN-I)via TLR9 (recognizing pathogenic DNA), whereas other cells are able toproduce low levels of IFN-I via different pathways, independent of TLR9.

Herein, it is described that some pathogenic poxviruses like Ectromeliavirus completely abolish the TLR9 independent IFN-I production offibroblasts and conventional cDC, whereas the TLR9 driven IFN-Iproduction of pDC is only reduced but not prevented. In vivo infectionstudies with the mouse specific poxvirus Ectromelia revealed that micelacking TLR9 have a more than 100-fold increase in susceptibility. Asimilar susceptibility and death kinetic in mice unable to respond toIFN-I was not found, which is known to be essential for fighting viralinfection. It is concluded that, under conditions where pathogenicviruses effectively inhibit the TLR9 independent recognition, theimportance of TLR9 dependent viral recognition and IFN-I productionbecomes essential. Thus, it is herein demonstrated that TLR9 is animportant, and in vivo highly relevant, PRR for the defense againstpoxviruses.

MVA-BN®, a highly attenuated poxvirus that has lost the ability toreplicate in mammals, is a potent inducer of robust adaptive immuneresponses, and vaccinated individuals are protected against speciesspecific poxviruses (e.g. Mousepox, monkeypox, Vaccinia). However,effective induction of adaptive immune responses takes several days toweeks, which leaves individuals unprotected against exposure to thesepathogens during that time. It is shown herein that MVA-BN® induces theproduction of innate immune-protecting cytokines (e.g. IFN-I),importantly via both TLR9 dependent and independent pathways. Thisproduction of innate immune-protecting cytokines by MVA is an unspecificimmune response, which can be employed in strategies to protect againsta multitude of pathogens.

Co-administration of MVA-BN® together with the highly pathogenicmousepoxvirus Ectromelia protected immune competent mice against dosesof Ectromelia at least 47-fold the lethal dose. In the absence of TLR9.MVA induced immediate protection to doses of Ectromelia at least500-fold the lethal dose. Experiments further demonstrate that even micethat lack responsiveness to IFN-I can be protected via MVAadministration. Since MVA protects, if applied at the same time as, orafter, the pathogenic poxvirus, the results described herein show animmediate protection, which is much earlier but in addition much morepronounced (>500×LD50) than previous data showing that MVA has to beapplied at the latest 2 days before the exposure to the pathogenic virusto gain some survival benefit (1×LD50) (WO2006/089690 and Staib et al.2006, J. Gen. Virol. 87:2917-2921 (2006)).

Administration of MVA around the time of lethal Ectromelia infection ledto a solid immediate protection against death in immune competent andTLR9 deficient mice. This immediate protection was only partiallydependent on a functional IFN-I pathway but fully dependent on adaptiveimmune responses, as shown with IFN-I-Receptor and Rag-1 deficient micerespectively. Importantly, MVA also rescued TLR9 deficient mice ifadministered two full days after an otherwise lethal infection withEctromelia virus. Thus, MVA induced a solid immediate and evenpost-exposure protection against lethal poxvirus infection inimmune-competent as well as immune-compromised animals.

MVA not only protects immediately, but the induced protection is, inaddition, long lasting. Mice lacking TLR9 (LD50=19) treated with only 1application of MVA were protected when challenged after 9 weeks withhigh lethal doses of Ectromelia (>500×LD50).

MVA-BN® not only induces strong adaptive immune responses (for examplehigh titers of neutralizing antibodies), but in addition induces stronginnate immune responses via cells including dendritic cells, thatproduce IFN-I which leads to a highly effective and, importantly,immediate protection against challenge with lethal poxvirus. Thus,MVA-BN® bridges innate and adaptive immune responses which results inimmediate and long lasting protection to lethal poxvirus challenge. Thisprotection can be extended to other pathogens by using a recombinant MVAexpressing antigenic epitopes of the pathogens.

In these studies, MVA not only protected if given around the time ofinfection, but application as late as 2 days fully and 3 days partiallyprotected mice from lethal ECTV infection. Prior scientific evidence forpost-exposure vaccination in orthopoxvirus-naïve individuals is lacking(Mortimer, P. P. 2003. Can postexposure vaccination against smallpoxsucceed? Clin. Infect. Dis. 36:622-629). The statements of officialHealth sites, e.g. WHO, of the potential success of post-infectionvaccinations most likely were with regard to individuals who hadpreviously been vaccinated against smallpox, thus referring to boostvaccination which most likely quickly enhanced existing adaptive memoryresponses. However, after the successful eradication of variola as anatural pathogen in the 1980's, widespread vaccinations were halted andnow the majority of the world population has never been vaccinatedbefore. Moreover, the prior studies were performed on patients who werevaccinated with a fully replication competent poxvirus, not MVA.

Animal models using either VACV-WR in mice or MPXV in monkeys have notshown therapeutic protection (Stittelaar et al., J. Virol. 79:7845-7851(2005); Staib et al., J. Gen. Virol. 87:2917-2921 (2006)).

Several reasons could explain the differences between the describedmodels and the present findings. Both VACV-WR in mice and MPXV inmonkeys are regarded as models reflecting only a late stage of smallpoxinfection due to the non-physiologically high doses which need to beapplied for a lethal infection of the respective model animals. However,ECTV lethal infections in mice can be induced with low virus doses viathe respiratory route, more resembling the beginning of a naturalsmallpox infection. Furthermore, VACV-WR induces pathologies in micesuch as high neurovirulence, drastic drop of body weight andtemperature, features not typical of ECTV in mice or VARV infections inhumans. This and possibly other reasons lead to a very rapid death ofthe infected animals, again not seen in the ECTV model or during naturalsmallpox infection.

In the case of the MPXV challenge in monkeys, therapeutic vaccinationwas done with VACV-Elstree. It was tested whether VACV-Elstree would beinhibitory and it was found that DC maturation and cytokine productionin vitro was inhibited as seen with other VACV strains. Given thattherapeutic protection presumably needs a solid induction of innateimmune mechanisms including antiviral cytokines to bridge the time forthe adaptive immune responses to develop, therapeutic application of anon inhibitory orthopoxvirus like MVA might be also beneficial inmonkeys infected with MPXV. Indeed, Staib and colleagues (2006) haveshown that mice were better protected with MVA than with VACV-Elstree ifapplied latest 2 days before challenge with VACV-WR (Staib et al., J.Gen. Virol. 87:2917-2921 (2006)). Thus, MVA seems to display protectiveadvantages over VACV-Elstree in pathogenic orthopoxvirus infectionmodels where the induction of innate immune mechanisms plays animportant role.

The invention induces robust and, most importantly, immediate protectionto a very high dose of exposure with a species-specific poxvirus innormal as well as immune compromised individuals. Moreover, thisprotection is long lasting. Thus, the invention provides an idealtreatment under conditions where quick protection against deadlypoxvirus infections is needed (e.g. terroristic or accidental exposureto smallpox or other pathogenic poxviruses).

The invention also encompasses the use of MVA and recombinant MVA asemergency tools against a large panel of other pathogens. The inventionfurther encompasses other attenuated viruses and bacteria as emergencytools against a large panel of pathogens. In addition, the inventioncould be employed for therapeutic intervention, giving the emergencytools, e.g. MVA, after exposure to the pathogen, e.g., smallpox.

The invention encompasses the use of a poxvirus for the preparation of avaccine for the rapid induction of a protective immune response in ananimal, including a human, wherein the poxvirus is replicationincompetent in the animal, including in the human.

The invention also encompasses a vaccine comprising a poxvirus for therapid induction of a protective immune response in an animal, includinga human, wherein the poxvirus is replication incompetent in the animal,including in the human.

In one embodiment, the invention encompasses a method for the rapidinduction of a protective immune response in an animal, including ahuman, comprising the step of administering to the animal, including thehuman, a poxvirus that is replication incompetent in the animal,including in the human.

The term “replication incompetent” means that the virus shows anamplification ratio of less than 1 in human cell lines, such as the celllines 293 (ECACC No. 85120602), 143B (ECACC No. 91112502), HeLa (ATCCNo. CCL-2) and HaCat (Boukamp et al., J. Cell Biol. 106(3): 761-71(1988)), under the conditions as outlined in Example 1 of WO 02/42480for some specific MVA strains and that the virus does not replicate inhumans and in the mice model as described in the WO 02/42480publication.

In one embodiment, the invention encompasses a use, vaccine or method asabove, wherein the protective immune response is generated in less than2 days.

In one embodiment, the poxvirus is a Modified Vaccinia virus Ankara(MVA), particularly MVA 575, MVA 572 and, preferably, MVA-BN®.

The invention also encompasses uses, vaccines or methods as above,wherein the virus is a cloned, purified virus. Particularly, theinvention encompasses viruses obtained in a serum free cultivationprocess.

In one embodiment, the poxvirus is administered in a dose of 10⁵ to5×10⁸ TCID₅₀. The poxvirus, in particular MVA can be administered, forexample by oral, nasal, intramuscular, intravenous, intraperitoneal,intradermal, intra-utero and/or subcutaneous application. In smallanimals the inoculation for immunization is preferably performedparenterally or nasally, whereas in larger animals or humans asubcutaneous, intramuscular or oral inoculation is preferred.

In the context of the present invention the term “animal” covers alsohuman beings. More generally, the animal is a vertebrate animal,preferably a mammalian animal including a human. Specific examples foranimals are pets such as dogs, cats, economically important animals suchas calves, cattle, sheep, goats, horses, pigs and other animal such asmice, rats. The invention may also be used for economically importantbirds such as turkeys, ducks, goose and hens if viruses are used thatare capable to infect the bird's cells but not capable of beingreplicated to infectious progeny virus in said cells. The term “domesticanimals” as used in the present description refers preferably tomammalian domestic animals, more preferably to dogs, cats, calves,cattle, sheep, goat, pigs, horses, deers.

Preferably, the immune response is a protective immune response againsta poxvirus infection, preferably, a smallpox infection. The protectiveimmune response can preferably protect against a dose of 1, 5, 10, 25,50, 100, 250, or 500 LD 50 of smallpox.

In one embodiment, the poxvirus is a recombinant poxvirus, preferably arecombinant MVA-BN®.

The poxvirus can comprise at least one heterologous nucleic acidsequence. The term “heterologous” as used in the present applicationrefers to any combination of nucleic acid sequences that is not normallyfound intimately associated with the virus in nature, Preferably, theheterologous nucleic acid sequence is a sequence coding for at least oneantigen, antigenic epitope, and/or a therapeutic compound. A“therapeutic compound” encoded by the heterologous nucleic acid in therecombinant virus can be, e.g., a therapeutic nucleic acid such as anantisense nucleic acid or a peptide or protein with desired biologicalactivity. The antigenic epitopes and/or the antigens can be antigenicepitopes and/or antigens of an infectious agent. The infectious agentscan be viruses, fungi, pathogenic unicellular eukaryotic or prokaryoticorganisms, and parasitic organisms. The viruses can be selected from thefamily of Influenza virus, Flavivirus, Paramyxovirus, Hepatitis virus,Human immunodeficiency virus, or from viruses causing hemorrhagic fever.The infectious agent can be bacillus anthracis.

The insertion of heterologous nucleic acid sequences is preferably intoa non-essential region of the virus genome. Alternatively, theheterologous nucleic acid sequence is inserted at a naturally occurringdeletion site of the viral genome (for MVA disclosed in PCT/EP96/02926).Methods how to insert heterologous sequences into the viral genome suchas a poxviral genome are known to a person skilled in the art.

According to a further embodiment the invention concerns the recombinantpoxvirus according to the present invention for use as vaccine ormedicament.

In one embodiment, the MVA virus is a strain characterized by having atleast one, two, or preferably three of the following advantageousproperties:

-   -   (i) capability of reproductive replication in vitro in chicken        embryo fibroblasts (CEF), but no capability of reproductive        replication in a human cell line, as in the human keratinocyte        cell line HaCaT, the human embryo kidney cell line 293, the        human bone osteosarcoma cell line 143B, and the human cervix        adenocarcinoma cell line HeLa;    -   (ii) failure to replicate in a mouse model that is incapable of        producing mature B and T cells and as such is severely immune        compromised and highly susceptible to a replicating virus; and    -   (iii) induction of at least the same level of specific immune        response in vaccinia virus prime/vaccinia virus boost regimes        when compared to DNA-prime/vaccinia virus boost regimes.

For the preparation of immunogenic compositions, the MVA vacciniaviruses according to the invention are converted into a physiologicallyacceptable form. This can be done based on the experience in thepreparation of MVA vaccines used for vaccination against smallpox (asdescribed by Stickl, H. et al., Dtsch. med. Wschr. 99:2386-2392 (1974)).Typically, about 10⁶-10⁸ particles of the recombinant MVA arefreeze-dried in phosphate-buffered saline (PBS) in the presence of 2%peptone and 1% human albumin in an ampoule, preferably a glass ampoule.The lyophilisate can contain extenders (such as mannitol, dextran,sugar, glycine, lactose or polyvinylpyrrolidone) or other aids (such asantioxidants, stabilizers, etc.) suitable for parenteral administration.The glass ampoule is then sealed and can be stored, preferably attemperatures below −20° C., for several months.

For administration or therapy the lyophilisate can be dissolved in 0.1to 0.5 ml of an aqueous solution, preferably physiological saline, andadministered either parenterally, for example by intramuscularinoculation. Immunogenic compositions, vaccines or therapeuticsaccording to the invention are preferably injected intramuscularly(Mayr, A. et al., Zbl. Bakt. Hyg., I. Abt. Orig. B 167:375-390 (1978)).The mode of administration, the dose and the number of administrationscan be optimized by those skilled in the art in a known manner. It isexpedient, where appropriate, to administer the immunogeniccompositions, vaccines or therapeutics one time, or several times over avariable period in order to obtain appropriate immune responses againstthe foreign antigen.

In one embodiment, the poxvirus is an inactivated orthopoxvirus.Preferably, the orthopoxvirus is inactivated with UV radiation. Inpreferred embodiments, the orthopoxvirus is CVA, ECTV, or CPXV.

It is an object of the present invention to provide a method forvaccinating an individual against a pathogen so as to provide immediateprotection. In one embodiment, the individual is vaccinated with MVA,preferably MVA-BN®, near the time of pathogenic exposure. Preferably,the vaccination is between 2 days prior to exposure and 3 dayspost-exposure. More preferably, the vaccination is between 2 days priorto the exposure and 1 day post-exposure. Even more preferably, thevaccination is between 1 day prior to the exposure and 1 daypost-exposure. The vaccination can be at 2 days prior, 36 hours prior, 1day prior, 12-24 hours prior, or 0-12 hours prior to the exposure. Thevaccination can also be at the time of the exposure or 0-12 hourspost-exposure, 12-24 hours post-exposure, 1 day post-exposure, 2 dayspost-exposure, 0-36 hours post-exposure, 0-48 hours post-exposure, 0-60hours post-exposure, 0-72 hours post-exposure, 3 days post-exposure, 4days post-exposure, or even 10 days post-exposure.

The invention includes a method for inducing a immune response againstan infectious agent in an animal comprising administering to the animalan immunogenic composition comprising an MVA, preferably MVA-BN®, at 2to 0 days, or 1 to 0 days, or any other combination of the hourscomprised by these days (e.g., 48-36, 48-24, 36-24, 24-12, 12-0, etc.)prior to infection with an infectious agent. In one embodiment, theinfectious agent is a replication competent poxvirus. In a preferredembodiment, the animal is a human.

The invention includes a method for inducing a immune response againstan infectious agent in an animal comprising administering to the animalan immunogenic composition comprising an MVA, preferably MVA-BN®, at 0to 3 days, 0 to 2 days, 0 to 1 days, or 1 to 2 days, or any othercombination of the hours comprised by these days (e.g., 0-12, 12-24,24-36, 24-48, 24-72, 36-48, 48-60, 48-72, etc.) after infection with aninfectious agent. In one embodiment, the infectious agent is areplication competent poxvirus. In a preferred embodiment, the animal isa human.

The invention further encompasses uses of the above methods and kitscomprising an immunogenic composition comprising an MVA, preferablyMVA-BN®, and instructions to deliver the immunogenic composition at atime point between 2 and 0 days prior to exposure to an infectiousagent, including 2, 1, or 0 days prior to exposure, as well as 36, 12,6, 3, or 1 hour prior to exposure. The time point can be within anycombination of the hours comprised by these days (e.g., 48-36, 48-24,36-24, 24-12, 12-0, etc.) prior to infection with an infectious agent.

The invention also encompasses uses of the above methods and kitscomprising an immunogenic composition comprising an MVA, preferablyMVA-BN®, and instructions to deliver the immunogenic composition at atime point between 0 and 3 days after exposure to an infectious agent,including 0, 1, 2, or 3 days after exposure, as well as 1, 3, 6, 12, 36,or 60 hours after exposure. The time point can be within any combinationof the hours comprised by these days (e.g., 0-12, 12-24, 24-36, 24-48,24-72, 36-48, 48-60, 48-72, etc.) after infection with an infectiousagent.

The invention also encompasses kits for the induction of a protectiveimmune response in an animal. Preferably, said immune response isdirected against an infectious agent as defined herein. In oneembodiment, the kit comprises an immunogenic composition comprising apoxvirus, wherein said poxvirus is replication incompetent in saidanimal. In a preferred embodiment, said poxvirus is a Modified VacciniaVirus Ankara (MVA). The kit may also comprise instructions for thedelivery of the immunogenic composition. The MVA is preferably MVA-BN.Preferably, the immunogenic composition contains 10⁵ to 5×10⁸ TCID₅₀/mlof MVA. In a preferred embodiment, the animal is a human. Theinstructions for delivery of the immunogenic composition can direct thedelivery at various time points prior to exposure or after exposure toan infectious agent. These time points can include time points between 2days prior to exposure to an infectious agent and 3 days after exposureto the infectious agent. In one embodiment, the instructions direct thatthe MVA is delivered after exposure to the infectious agent, preferablyas soon as possible after exposure to the infectious agent, which ispreferably smallpox. The kit may further comprise an infectious agent asdefined herein in a separate vial and instructions for the delivery ofthe infectious agent to an animal, including a human. The infectiousagent is preferably selected from bacillus anthracis or smallpox.

In this context, an “exposure” means contact with the infectious agentitself, or with an animal (human) harboring the infectious agent. Forexample, the instructions can direct that the immunogenic compositioncan be delivered at 36, 24, 12, 6, 3, or 1 to 0 hours prior to exposureto an infectious agent or at 0 to 1, 3, 6, 12, 24, 36, 48. 60, or 72hours after exposure to an infectious agent. For example, theinstructions can direct delivery at 48-36, 48-24, 36-24. 24-12, 12-0,etc., prior to infection with an infectious agent or 0-12, 12-24, 24-36,24-48, 24-72, 36-48, 48-60, 48-72, etc. after infection with aninfectious agent. Preferably, the infectious agent is smallpox orbacillus anthracis. The instructions can direct that the MVA beadministered intravenously, intramuscularly, and/or subcutaneously. Theinstructions can direct that the MVA be administered intranasally.

The pathogen is preferably a virus or a bacterium. In a preferredembodiment, the pathogen is a poxvirus, preferably a Variola virus.

In one embodiment, the individual is a healthy human. In anotherembodiment, the individual is an immunocompromised human, for example,an HIV-1 infected individual, an individual with atopic dermatitis, apatient taking immunosuppressive drugs, or an individual with allergies.

Modified vaccinia virus Ankara (MVA), a host range restricted and highlyattenuated vaccinia virus strain, is unable to multiply in human andother mammals tested. But, since viral gene expression is unimpaired innon-permissive cells, the recombinant MVA viruses according to theinvention may be used as exceptionally safe and efficient expressionvectors.

Poxviruses including the causative agent of smallpox, variola virus,have developed multiple strategies to suppress immune responses. Theinvention provides evidence that poxviruses are recognized via toll-likereceptor (TLR)9-dependent as well as TLR9-independent pathways.Pathogenic poxviruses effectively suppressed their recognition via theTLR9-independent pathway employed by conventional dendritic cells (DC),but were detected by plasmacytoid DC (pDC) via TLR9. The lack of TLR9abrogated the DC response in vitro and drastically increased thesusceptibility of mice to infection with the murine poxvirus Ectromeliavirus (ECTV). Simultaneous administration of modified vaccinia virusAnkara (MVA)-BN® at the time of infection led to a solid immediateprotection against ECTV, even in the absence of TLR9 or interferon typeI receptor (IFN-I-R). MVA-BN® also rescued mice if administered afterinfection with ECTV. Thus, MVA-BN® induced a solid immediate and evenpost-exposure protection against lethal ECTV infection inimmune-competent as well as immune-compromised mice.

The data presented below in Examples 1 through 11 demonstrate thatpoxviruses, as shown previously for other families of dsDNA viruses, aredetected via TLR9-dependent as well as TLR9-independent recognitionpathways. MVA, a highly attenuated VACV that has lost its capacity toreplicate in human cells, was found to be recognized by pDC via bothTLR9-dependent and TLR9-independent pathways, whereas in cDC it was onlyrecognized via the TLR9-independent pathway. This finding is consistentwith previous findings with HSV-1 (Hochrein, H. et al., Proc. Natl.Acad. Sci. U.S.A 101, 11416-11421 (2004)). However, in sharp contrast,the recognition of the pathogenic poxviruses, including several strainsof VACV, ECTV or CPXV critically relied on TLR9 and pDC, likely due tothe potent ability of these viruses to inhibit their recognition viaTLR9-independent pathways. In the absence of pDC or TLR9, thisinhibitory potential nearly completely abrogated immune recognition andthus response by DC in vitro. This translated into the in vivo infectionmodel with ECTV, where TLR9 deficient mice were more than 100-fold moresusceptible than wild type mice.

Since the response to the inactivated VACV, CPXV and ECTV virusesdepended on the presence of TLR9, these viruses most likely inhibit boththe TLR9-dependent and the TLR9-independent activation pathways.Strikingly, in the absence of TLR9, this inhibition was virtuallyabsolute as even the most sensitive readout of DC maturation (CD69expression) was abolished. Several poxvirus encoded inhibitory genesincluding the VACV product of A46R and A52R (Bowie et al., Proc. Natl.Acad. Sci. U.S. A. 97:10162-10167 (2000); Harte et al., J. Exp. Med.197:343-351 (2003); Stack et al., J. Exp. Med. 201:1007-1018 (2005))have been implicated in the inhibition of TLR signaling molecules.Genetic comparisons of ECTV, CVA, CPXV and MVA show that all 4 viruseshave homologues of A46R (Meisinger-Henschel et al., J. Gen. Virol.88:3249-3259 (2007)). CVA and CPXV also express homologues of A52Rwhereas MVA lacks this component. ECTV strain Moscow which we have usedfor this study has a fragmented A52R gene which most likely is notfunctional (Chen et al., Virology 317:165-186 (2003)). To elucidate thepotential role of A46R and A52R in the inhibition of DC activation viathe TLR9 dependent and independent recognition pathways, recombinantVACVs lacking or expressing A46R and A52R have been constructed. Arecombinant MVA expressing A52R as well as endogenous A46R, thusresembling the inhibitory CVA (endogenously expressing A46R and A52R),did not demonstrate any significant increased inhibition compared towild type MVA as judged by analyses of DC maturation and cytokineinduction. In contrast, a deletion mutant of CVA expressing neither A46Rnor A52R did not lose its inhibitory potential. This is in accordancewith previously published data showing that an A52R defective VACV stillretained inhibitory activity against DC maturation (Drillien et al, J.Gen. Virol. 85:2167-2175 (2004)). Taken together, these data suggestthat neither A46R (endogenously expressed by the non inhibitory MVA) norA52R are the major inhibitory component of the TLR9-dependent orTLR9-independent recognition defined within this study.

The nature of TLR9-independent recognition in response to MVA is stillelusive as are the recognition pathways in response to other DNA viruses(Ishii et al., Trends Immunol 27:525-532 (2006)). However, recentreports rule out the absolute dependence on the presence of the TLRassociated adaptor molecules MyD88 and TRIF as well as PKR (Zhu et al.,Blood 109:619-625 (2007); Waible et al., J. Virol. 81:12102-12110(2007)). Recently a new intracellular sensor for DNA (DAI) wasidentified (Takaoka et al., Nature 448:501-505 (2007)). Infecting cellsin the presence or absence of a siRNA silencing DAI indicated that theresponse to transfected DNA or HSV-1 but not to RNA was to some extentdependent on DAI. However, the response to HSV-1 was reduced but notabrogated (responses to poxviruses were not tested) suggesting theexistence of additional DNA virus recognition pathways. Others haveshown that mouse embryonic fibroblasts responded to MVA (lacking thegene E3L) independently of the presence of the noncanonical IKB kinasefamily members TBK1 and IKKi (Ishii et al., Nat. Immunol. 7:40-48(2006)) and that the induction of IFN-α in response to MVA wasindependent of virus propagation and DNA replication (Waible et al., J.Virol. 81:12102-12110 (2007)).

In the case of TLR9-independent recognition of HSV, recent publicationssuggest that different cell types might have different requirement. IncDC, the IFN response was independent of viral replication but dependenton viral entry. In contrast, in macrophages and fibroblasts, IFN-Iproduction was dependent on both viral entry and replication and inaddition on a functional mitochondrial signaling protein pathway, whichsuggest a possible involvement of RNA components (Rasmussen et al., J.Virol. 81:13315-13324 (2007); Weber et al., J. Virol. 80:5059-5064(2006)). Thus, the immunological recognition of DNA viruses seems atleast as redundant as the recognition of RNA viruses. Suppressivemechanisms developed by different viruses, some of those employed bypoxviruses, most likely put an enormous evolutionary pressure on thedevelopment of redundant DNA virus recognition pathways.

Poxviruses are divided into two subfamilies, the poxviruses infectinginvertebrates e.g. insects (entomopoxvirinae) and poxviruses infectingvertebrates (chordopoxvirinae). Many, if not all species of vertebrates,have battled throughout evolution for their survival against highlypathogenic poxviruses. Today, poxviruses infecting reptiles, birds andmany different species of mammals are known. Vertebrates as early asfishes are known to respond to CpG-DNA stimulation suggesting theexpression of TLR9. One could speculate that the TLR9 system, as well asthe specialized DC subset employing TLR9 for IFN-I production, pDC, wereoptimized under strong evolutionary pressure for the detection of andthe defense against poxviral infections.

TLR9 expression in murine and human cells differs greatly. Whereas inboth species pDC and B-cells are positive for TLR9 and respond to TLR9stimulation, murine cDC subsets and macrophages also express TLR9.Moreover, different cell types even within one species responddifferentially and selectively to TLR9 ligands. This includes not onlythe unique IFN-α producing capacity of pDC but is also demonstrated inselective responses of B-cells to different TLR9 agonists. Previously,it was described that murine B-cells are activated and proliferate to aB-type CpG-ODN but not to an A-Type CpG-ODN or to purified plasmid DNA(Spies et al., J. Immunol. 171:5908-5912 (2003)). One possibleexplanation could be cell type specific uptake and endosomal processingof different TLR9 ligands. This could also possibly explain why the cDCemployed in this study only displayed the TLR9-independent, but not anyTLR9-dependent stimulation in response to MVA, even though they expressTLR9 and respond to the artificial TLR9 agonist CpG-ODN.

The immune protection induced by MVA was dearly relevant in a settinglacking TLR9 responses and thus immune activation in response to MVA wasnot solely dependent upon TLR9 or pDC. This feature of MVA to induce pDCand IFN-I independent immune activation could be important underconditions described in humans where pDC numbers or function and thusTLR9-dependent IFN-I production are impaired. Among these are cancer andtransplantation patients, people taking immune suppressive drugs andpeople with HIV, even under antiviral treatment ((Hashizume et al., J.Immunol. 174, 2396-2403 (2005); Donaghy, H. et al., Blood 98, 2574-2576(2001); Chehimi, J. et al., J. Immunol. 168, 4796-4801 (2002); Boor, P.P. et al., Am. J. Transplant. 6, 2332-2341 (2006); Siegal, F. P. et al.,J. Clin. Invest 78, 115-123 (1986)). Furthermore, some immune conditionslike allergies are associated with reduced virus induced IFN-Iproduction (Bufe et al., Int. Arch. Allergy Immunol. 127:82-88 (2002)).Of note, most of these conditions have been defined as contraindicatedfor the application of replication-competent smallpox vaccines.

Although we found that MVA was able to protect to some extent IFN-I-Rdeficient mice against ECTV infection (FIG. 7), application of thetraditional smallpox vaccine virus, Dryvax, killed these mice evenwithout ECTV challenge. This finding was consistent with previousreports on lethality to VACV Wyeth in other immune compromised mice(Wyatt et al., Proc. Natl. Acad. Sci. U.S.A. 101:4590-4595 (2004);Perera et al., J. Virol. 81:8774-8783 (2007)).

This study demonstrates that MVA given at the same time as lethal dosesof ECTV protected wild type and TLR9 deficient mice against death (FIG.4, FIG. 5) irrespective of the site of application, (FIG. 6), theprotocol called “immediate protection.” These findings indicate that MVAinduces solid innate immune responses and thus bridges the time adaptiveimmune responses need to develop.

To define the mechanisms of the innate protection phase, the immediateprotection protocol was applied to mice which lack responsiveness toIFN-I. Upon high dose exposure, these mice were not protected to thesame extent as wild type mice, suggesting that IFN-I is part of theprotection. However, IFN-I-R mice were protected to lower, butnevertheless lethal, doses of ECTV, clearly demonstrating that othermechanisms are able to substitute for IFN-I during the innate phase ofthe immediate protection protocol.

A role for TNF-α in protection against ECTV was previously shown by theincreased susceptibility of TNF-Receptor deficient mice to ECTVinfection as well as by the attenuation of TNF-α encoding VACV (Ruby etal., J. Exp. Med. 186:1591-1596 (1997)). Using similar methods,antiviral activities were reported for IL-2, IL-6, IL-12, IFN-gamma,IFN-lambda, CD40L, Mig, IP-10, NO and complement (Esteban et al., J.Gen. Virol. 86:2645-2659 (2005); Ramshaw et al., Immunol. Rev.159:119-135 (1997); Bartlett et al., J. Gen. Virol. 86:1589-1596 (2005);Niemialtowski et al., Acta Virol. 38:299-307 (1994)). Apart from solublecomponents, cellular innate mechanisms like the induction of NK cellsseem to play an important role during infections with poxvirusesincluding ECTV infection (Parker et al., J. Virol. 81:4070-4079 (2007)).These and other mechanisms may be involved in the MVA mediated immediateprotection.

The failure of the immediate protection protocol to induce sustainedprotection in the absence of adaptive immune responses (FIG. 8) clearlyindicated that survival depended ultimately on adaptive immuneresponses. The prolongation of survival in the RAG-1 deficient mice alsogave some indication of the duration of solid protecting innatemechanisms but as described previously for traditional vaccinationstrategies, survival to pathogenic poxvirus infection ultimately needsadaptive mechanisms to clear the virus. This prerequisite makes itunlikely that the sole induction of innate mechanisms like applicationof IFN-I, TLR ligands or other non-specific innate stimuli would sufficein the protection to lethal poxvirus infection if adaptive immuneresponses were not effectively triggered at the same time. Theexperiment with UV-inactivated CVA (FIG. 6 b) which carriesorthopoxvirus antigens and presumably activates via TLR9 suggested thatsome limited protection could be achieved in immune competent mice.However, the fact that all mice at least became sick, in stark contrastto the mice treated with active MVA which stayed symptom free, indicatedthat the protection via the active MVA is much more solid.

TLR9 was identified as an essential and in vivo highly relevantrecognition molecule for poxviruses. Importantly, it provides evidencefor the use of MVA-BN® as a way for immediate and therapeuticintervention against potential fatal poxvirus infection in healthy aswell as immune compromised individuals.

Here, previous data that members of the poxvirus family are recognizedvia TLR-independent recognition pathways (Zhu, J. et al. Blood 109,619-625 (2007), are confirmed. However, it is shown that poxviruses arealso seen via the TLR9-dependent pathway. It is shown that somepoxviruses, like ECTV, effectively suppress the recognition via theTLR9-independent pathways but are still recognized via TLR9.

Plasmacytoid DC (pDC) are the only cells in human and mouse whichproduce large amounts of type I interferon (IFN-I) via the TLR9 pathway,whereas other cells including conventional DC (cDC) are able to produceIFN-I via different pathways, independent of TLR9. It is shown hereinthat some poxviruses completely abolish the TLR9-independent IFN-Iproduction and affect the maturation of DC, whereas the TLR9-drivenIFN-I production of pDC is not fully prevented.

In vivo studies with ECTV, a natural mouse pathogen, revealed that thelack of TLR9 renders mice more than 100 fold more susceptible toinfection. A similar susceptibility and death kinetics could be found inmice unable to respond to IFN-I, which is thought to be essential tocontrol viral infection (Muller, U. et al. Science 264, 1918-1921(1994)). Thus, under conditions where pathogenic DNA viruses effectivelyinhibit their TLR9-independent recognition, the roles of TLR9-dependentviral recognition, IFN-I production and thus pDC become critical atleast for primary defense mechanisms during infection.

MVA, a highly attenuated orthopoxvirus that has lost the ability toreplicate in mammals, is a potent inducer of robust adaptive immuneresponses and vaccinated individuals are protected against otherpoxvirus species within the genus Orthopoxvirus (e.g. monkeypox virus(MPXV)) (Earl, P. L. et al. Nature 428, 182-185 (2004); Stittelaar, K.J. et al. J. Vir. 79, 7845-7851 (2005)). Due to an inability toreplicate in mammals, MVA is tested as a vaccine candidate even inhighly immune compromised individuals (Gherardi et al., J. Gen. Virol.86:2925-293 6; Staib et al., J. Gen. Virol. 87:2917-2921 (2006)).However, effective induction of adaptive immune responses takes severaldays to weeks and previous reports have shown that limited survivalbenefits against pathogenic poxviruses can only be achieved withapplication of the vaccinating virus at the latest two days beforeexposure to the challenge virus (Staib, C. et al. J. Gen. Virol. 87,2917-2921 (2006)).

It is shown here that MVA-BN induces the production of innateimmune-protecting cytokines (e.g. IFN-I) in vitro via bothTLR9-dependent and -independent pathways. Unlike poxviruses such asECTV, MVA did not inhibit the ability of DC to recognize it viaTLR9-independent pathways. This property can be useful in protectionagainst poxviruses that displayed a more inhibitory phenotype.

Administration of MVA-BN at the same time as high doses of the highlypathogenic and species-specific mousepox virus ECTV protected not onlyimmune-competent mice against death, but also mice which lacked TLR9 orresponsiveness to type I interferon. Mice without a functional IFN-Iresponse were protected to low and intermediate ECTV challenges howeversuccumbed to infection if higher doses were used indicating that onemechanism of protection involves IFN-I which can be substituted to someextent by other means. However, mice lacking adaptive immune responses(Rag-1-deficient mice) had only some temporary advantage with MVAadministration, but all mice died finally indicating that the inductionof adaptive immune responses are essential for the overall protection tolethal poxvirus infection. Thus, MVA was capable of activating an immuneresponse in vivo via TLR9-independent pathways, even in the presence ofa poxvirus that potently inhibited this recognition. Importantly, evenpost-exposure application of MVA-BN protected TLR9-deficient mice fromdeath against lethal infection by ECTV.

This study demonstrates that TLR9 is an important, and in vivo highlyrelevant, PRR for the recognition of, and the defense against,poxviruses. Moreover, even under conditions of compromised immunesystems, MVA-BN activates and bridges innate and adaptive immuneresponses, resulting in long lasting but importantly also immediate andtherapeutic protection against lethal poxvirus challenge.

The data presented clearly demonstrate that poxviruses, as shownpreviously for other families of dsDNA viruses, are detected viaTLR9-dependent as well as TLR9-independent recognition pathways. MVA, ahighly attenuated VACV that has lost its capacity to replicate in humancells, was found to be recognized by pDC via both TLR9-dependent andTLR9-independent pathways, whereas in cDC it was only recognized via theTLR9-independent pathway. After UV-inactivation of MVA a mixedpopulation consisting of pDC and cDC produced cytokines only in thepresence of TLR9 (FIG. 2 b). This finding closely resembled our previousfindings with HSV-1 where active virus induced IFN-α in vitro in severalDC subsets and macrophages independent of TLR9 whereas pDC employed inaddition a TLR9-dependent pathway, that also recognized inactivated HSV(Hochrein et al., Proc. Natl. Acad. Sci. U.S.A. 101:11416-11421 (2004)).The inactivation methods employed (heat inactivation in the case ofHSV-1 and strong UV-irradiation in the case of MVA) potentially resultedin selective uptake of the viruses into different cellular compartments(active virus into the cytosol and the endo some whereas uptake ofinactivated virus might be restricted to the endosomal route). Thiscould be an explanation for the complete TLR9 dependence afterinactivation.

However, in sharp contrast to MVA, it is shown here that the recognitionof the pathogenic poxviruses, including several strains of VACV, ECTV orCPXV critically relied on TLR9 and pDC due to the potent ability ofthese viruses to inhibit their recognition via TLR9-independentpathways. In the absence of pDC or TLR9, this inhibitory potentialnearly completely abrogated immune recognition and thus response by DCin vitro. The in vitro findings translated into the in vivo infectionmodel with ECTV, where TLR9-deficient mice were more than 100-fold moresusceptible than wild type mice (FIG. 3). Other dsDNA virus infectionmodels in TLR9-deficient mice have shown either no increase ofsusceptibility as in the case of HSV-1 infections, or only moderateincreases within a narrow range in the case of MCMV infections (Krug etal., Blood 103:1433-1437 (2004); Tabeta et al., Proc. Natl. Acad. Sci.U.S.A. 101:3516-3521 (2004); Delale et al., J. Immunol. 175:6723-6732(2005)).

This study defines TLR9 as an important, and in vivo highly relevant,recognition molecule for poxviruses. Importantly, it provides evidencefor the use of MVA-BN as a way for immediate and therapeuticintervention against potential fatal poxvirus infection in healthy aswell as immune compromised individuals.

It is a further object of the present invention to use a recombinantpoxvirus, including, but not limited to an MVA virus, which can serve asan efficient and exceptionally safe expression vector. In oneembodiment, the present invention relates to recombinant MVA viruseswhich contain a gene which codes for a foreign antigen, preferably of apathogenic agent, and vaccines containing such a virus in aphysiologically acceptable form. The invention also relates to methodsfor the preparation of such recombinant MVA vaccinia viruses orvaccines, and to the use of these vaccines for the prophylaxis ofinfections caused by such pathogenic agents.

The MVA viruses according to the invention can also be recombinant MVAexpressing heterologous polypeptides. A DNA construct, which contains aDNA-sequence which codes for a foreign polypeptide flanked by MVA DNAsequences adjacent to a naturally occurring deletion, e.g. Deletion IIor an IGR, within the MVA genome, can be introduced into cells infectedwith MVA, to allow homologous recombination. Once the DNA construct hasbeen introduced into the eukaryotic cell and the foreign DNA hasrecombined with the viral DNA, it is possible to isolate the desiredrecombinant vaccinia virus in a manner known per se, preferably with theaid of a marker (compare Nakano et al., Proc. Natl. Acad. Sci. USA,79:1593-1596 (1982); Franke et al., Mol. Cell. Biol, 1918-1924 (1985);Chakrabarfi et al., Mol. Cell. Biol., 3403-3409 (1985); Fathi et al.,Virology 97-105 (1986)).

In one embodiment, in immune competent mice MVA immediately protectsmice against the mousepoxvirus Ectromelia (>47×LD50).

In one embodiment, MVA induces immune responses in dendritic cells viaTLR9 and in addition via TLR9-independent pathways. Pathogenicpoxviruses like Ectromelia virus in contrast inhibit effectively theTLR9-independent recognition and thus depend on TLR9 for recognition.

In one embodiment, the immune compromised mice lacking TLR9 (TLR9-KO)have a 100-fold higher susceptibility to Ectromelia infection.

In one embodiment, MVA immediately protects TLR9-KO mice against themouse poxvirus Ectromelia (>500×LD50).

In one embodiment, MVA protects immune compromised mice (lackingresponsiveness to IFN-I) against low to intermediate challenge withEctromelia virus (24 of 25 mice survived an otherwise deadly exposure toEctromelia with 1E+02 or 1E+03).

In one embodiment, protection achieved by only one application of MVA islong-lasting. After 9 weeks, there is still protection against firsttime infection with Ectromelia (>500×LD50).

The detailed examples which follow are intended to contribute to abetter understanding of the present invention. However, the invention isnot limited by the examples.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

EXAMPLES

The following examples will further illustrate the present invention. Itwill be well understood by a person skilled in the art that the providedexamples in no way may be interpreted in a way that limits theapplicability of the technology provided by the present invention tothese examples.

Example 1 Experimental Methods

The following section is a summary of those methods used in all of theExamples described herein.

Animal Model

C57BLJ6J mice were purchased from Harlan Winkelmann (Borchen, Germany).TLR9 deficient mice were generated with 129/Sv background andbackcrossed to C57BL/6 for at least 8 generations as described (Hemmi,H. et al., Nature 408, 740-745 (2000); Hochrein, H. et al.). Both the129/Sv as well as C57BL/6 mouse strains are regarded to display arelatively high resistance to ECTV infection (Tscharke et al., J. Exp.Med. 201:95-104 (2005)). However, to rule out that in the infectionmodel the strain background would have an influence mice on the 129/Svbackground were infected with ECTV i.n. and it was found that indeedthey displayed the relative resistant phenotype seen in C57BLJ6 mice,e.g. none of the mice died with the dose of 1E+02 TCID50 and themajority of mice even survived a dose of 3E+03. IFN-I-R deficient mice(A129) mice were originally obtained from Dr. Michel Aguet (Universityof Zurich) (Muller, U. et al., Science 264, 1918-1921 (1994)) andbackcrossed to C57BL/6 mice for 8 generations. RAG-1 deficient mice werepurchased from the Jackson laboratories and bred at the animal facilityin Zurich.

Viruses

The MVA used for this study was MVA-BN®, developed by Bavarian Nordicand deposited at European Collection of Cell Cultures (ECACC)(V00083008). MVA was propagated and titered on primary chicken embryofibroblasts (CEF) that were prepared from 11-day-old embryonatedpathogen-free hen eggs (Charles River, Mass., USA) and cultured inRPMI-1640 medium supplemented with 10% FCS. CVA and CNPV were kindlyprovided by Prof. A. Mayr, Veterinary Faculty, Munich, Germany and werepropagated and titered on CEF. ECTV strain Moscow and CPXV strainBrighton were obtained from the American Type Culture Collection (ATCC)as VR-1372 and VR-302, respectively, and were propagated and titered onVero C1008 cells (ECACC 85020206). SFV was obtained from ATCC (VR-364)and propagated and titered on the rabbit cornea cell line SIRC obtainedfrom ATCC (CCL-60).

All cell lines were maintained in Dulbecco's Modified Eagle's Medium(DMEM; Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS withoutantibiotics. All viruses used in animal experiments were purified twicethrough a sucrose cushion. For the UV-inactivation of virusesconcentrated virus stocks were UV irradiated with an UV Chamber(Genelinker GS, Bio-Rad laboratories, Munich Germany) for 15 min understerilizing conditions. This treatment reduced the transductionefficiency of recombinant viruses below 2% of the original virusactivity.

In Vitro Experiments

In vitro generated Flt3-ligand-dependent DC (FL-DC) were generated andsorted essentially as described previously (Hochrein, H. et al., Proc.Natl. Acad. Sci. U.S.A. 101, 11416-11421 (2004)). In short, bone marrowcells were cultured in the presence of murine recombinant FL for eightdays. Resulting cells were >90% CD1 1c positive and 20-60% of cellsdisplayed plasmacytoid phenotype (CD11c^(pos)CD45RA^(high)B220^(high)CD1 1b^(low)). FL-DC were either usedunseparated or sorted into pDC and cDC using a FACS Aria instrument (BDBioscience). In vitro generated GM-DC were generated by culturing bonemarrow cells in the presence of murine recombinant GM-CSF (Tebu-bio,Offenbach, Germany) as described (Hochrein, H. et al., Proc. Natl. Acad.Sci. U.S.A. 101, 11416-11421 (2004)). Cells were stained with antibodiesspecific for CD1 1c, B220, CD40 and CD69 (BD Biosciences). Propidiumiodide (1 μg/ml) was included in the final wash to label dead cells.Flow cytometric analyses were performed on a FACSCalibur (BD Bioscience)and analyzed with Weasel software (The Walter and Eliza Hall Institutefor Medical Research, Melbourne, Australia). Cell culture supernatantswere harvested 18-24 hours after incubation with the viruses asindicated or with CpG-2216 (0.5 μM or 1 μM) as control in the presenceof IL-3 and GM-CSF and the secretion of IFN-└ and IL-6 was measuredusing commercially available ELISA-reagents as described previously(Hochrein, H. et al. (2004)).

In Vivo Experiments and Statistics

Mice were anaesthetized with ketamine/xylamine and viruses were appliedby i.n. drop wise installation in a total volume of 50 μl.ECTV-dilutions as indicated were applied either alone or in combinationwith 1E+08 TCID₅₀ MVA. Subcutaneous injections were performed in theinguinal region applying a total amount of 1E+08 TCID₅₀ of MVA or acorresponding amount of UV-inactivated CVA by injecting 2 times a volumeof 250 μl each. The health status of infected mice was checked at leastdaily and animals with grave symptoms of sickness or weight lossexceeding 25% were euthanized. For the determination of poxvirusspecific CD8⁺T cell responses wild type or TLR9-deficient mice wereinfected intravenously with 5E+07 TCID₅₀ or 1E+08 TCID₅₀ MVA. Spleenswere harvested 7 days after immunization and single cell suspensionswere prepared by mechanically disrupting the organs through a 70-μmfilter. Spleen cells and peripheral blood lymphocytes (PBL) were treatedwith red blood cell lysis buffer (0.14 M NH₄Cl and 0.017 M Tris-HCI, pH7.2), washed twice, and analyzed. Cells were stained with Pro5® H-2 KbPentamers (Prolmmune, Oxford, UK) loaded with the immunodominant B8Rpeptide TSYKFESV (Tscharke et al., J. Exp. Med. 201:95-104 (2005)).Pentamer staining was performed in combination with anti-CD8, anti-CD 19and anti-NK1 1 antibodies according to the manufacturers' protocol. Forintracellular cytokine staining cell suspensions were stimulated for 5hrs with 1 μg/ml B8R peptide in the presence of 1 μg/ml GolgiPlug (BDBiosciences). Afterward, cells were surface stained with anti-CD8 andthen simultaneously fixed/permeabilized with the BD Cytofix/Cytoperm Kit(BD Biosciences) and finally stained with antibodies directed againstIFN-α, TNF-I and IL-2. Poxvirus specific antibodies in sera weremeasured by ELISA using MVA crude extract as antigen and asheep-antimouse-IgG-HRP (Serotec, Germany) as detection antibody. Allanimal experiments were approved by the government of Bavaria. For thecalculation of the _(LD50), the Spearman-Karber method was used.

Example 2 Inactivation of VACV, CPXV and ECTV but not of MVA, CNPV andSFV Increases DC Maturation

Previously it has been described that several strains of VACV inhibitthe maturation of cDC whereas maturation occurred in response to MVA(Engelmayer et al., J. Immunol. 163:6762-6768 (1999); Drillien et al.,J. Gen. Virol. 85:2167-2175 (2004)). Since these studies analyzed onlythe role of cDC in the absence of pDC Flt3-ligand (FL)-generated murineDC were employed, that consist of DC that closely resemble ex-vivo mousespleen cDC and pDC (Naik et al., J. Immunol. 174:6592-6597 (2005)), toexamine the activation of both DC types. To test whether differentstimulatory activities of different VACV were due to lack of stimulus oractive inhibition by a virus-encoded component, FL-DC were incubatedwith several different strains of poxviruses either as active virus orafter UV-inactivation. The activation of DC in response to VACV strainAnkara (CVA), ECTV and CPXV was amplified after viral UV-inactivationcompared to the activation of DC in response to active viruses (FIG. 1a). These initial data indicated an inhibitory component, acting on DC,was made by those viruses. In contrast, DC activation as measured by theupregulation of CD40, CD69 and CD86 in response to MVA as well as thecanarypox virus (CNPV) and rabbit Shope fibroma virus (SFV) was notincreased after UV-inactivation (FIG. 1 b and data not shown),suggesting that these viruses lacked an active inhibitory component.Apart from DC maturation, the production of cytokines including IFN-αand IL-6 increased after the UV-inactivation of CVA, ECTV and CPXV butnot of MVA, CNPV and SFV, suggesting a broad inhibition of viralrecognition and DC function, not restricted to maturation.

Example 3 Recognition of CVA and ECTV but not of MVA Exclusively Dependson TLR9

It has previously been shown that dsDNA viruses like herpesviruses oradenoviruses could be recognized via TLR9-dependent as well asTLR9-independent recognition pathways (Basner-Tschakarjan et al., J.Gene Med. 8:1300-1306 (2006); Hochrein et al., Proc. Natl. Acad. Sci.U.S.A. 101:11416-11421 (2004)). To elucidate the role of TLR9 in therecognition of poxviruses FL-DC of wild type or TLR9-deficient animalswere generated. In the absence of TLR9, DC did not significantly maturein response to active CVA or ECTV, as monitored by the lack ofupregulation of CD40 and CD69, indicating a strong dependence upon TLR9for the recognition of these viruses. However, in the absence of TLR9MVA induced robust upregulation of CD69 but a drastically reducedupregulation of CD40 (FIG. 2 a), suggesting that the response to MVA isbased on both TLR9-independent and TLR9-dependent recognition events.

FL-DC contain both pDC, known as the sole cell type producing largeamounts of IFN-α in response to TLR9 activation, and cDC, known to beunable to produce large scale IFN-α production in response to TLR9.FL-DC of wild type and TLR9-deficient mice were incubated with activeMVA and produced dose-dependent robust amounts of IFN-α and IL-6demonstrating the existence of a TLR9-independent recognition pathwayfor MVA (FIG. 2 b). However, UV-inactivated MVA induced IFN-α and IL-6solely in wild type but not in TLR9 deficient FL-DC, reinforcing thenotion suggested by the maturation data, that the recognition of MVAalso employed a TLR9-dependent component (FIG. 2 b).

DC generated in vitro with GM-C SF resulted in a DC population (GM-DC)of only cDC which are able to produce IFN-α in response to active DNAviruses (e.g. Herpes simplex virus (HSV)) but not to inactivated virusesor CpG-ODN (Hochrein et al., Proc. Natl. Acad. Sci. U.S.A.101:11416-11421 (2004)). Incubation of GM-DC with active MVA inducedIFN-α and IL-6 production in wild type and TLR9-deficient cells,demonstrating the TLR9-independent recognition of active MVA. No IFN-αproduction and no IL-6 above constitutive levels produced was detectedafter incubation with UV-inactivated MVA (FIG. 2 c) potentiallyindicating that the TLR9-dependent recognition in response to MVA is notfunctional in those cells.

Example 4 Recognition of ECTV but not of MVA Exclusively Depends on TLR9and pDC

To define the individual activation profiles of the two main DC subsetsamong the FL-DC pDC and cDC were sorted, infected with ECTV and MVA, andthe IFN-α and IL-6 production was measured. Wild type pDC produced IFN-αto both ECTV and MVA and very little IL-6 to ECTV. However cDC orTLR9-deficient pDC only produced IFN-α in response to MVA but not ECTV(FIG. 2 d). Wild type and TLR9-deficient cDC also produced large amountsof IL-6 in response to MVA but not to ECTV. These results strengthen theobservations obtained with DC maturation (FIG. 2 a) and demonstrate thateffective recognition of ECTV depends on the presence of TLR9, and inparticular that IFN-α production by ECTV is dependent upon pDC. ECTVclearly inhibits recognition via other TLR9-independent pathways. On theother hand, recognition of MVA by both pDC and cDC is composed of anadditional TLR9-independent mechanism.

To analyze ex vivo isolated pDC containing cell populations in additionto in vitro generated FL-DC, wild type and TLR9-deficient total bonemarrow cells, a rich source of pDC in vivo, were stimulated with activeor UV-inactivated MVA in parallel to CpG-ODN as a control. Similar toresults with FL-DC, active MVA induced robust IFN-α production in wildtype and TLR9-deficient bone marrow cells, whereas with the lack of TLR9the IFN-α production in response to UV-inactivated MVA and CpG-ODN wascompletely abrogated (FIG. 2 e). Thus, these data demonstrated that MVAwas recognized by freshly isolated bone marrow cells via a UV-sensitiveTLR9-independent pathway and a TLR9-dependent pathway.

Example 5 TLR9-Deficient Mice have a Drastically IncreasedSusceptibility to ECTV Infection

Previous reports have clearly demonstrated the recognition of DNAviruses by TLR9 in vitro but the in vivo relevance of TLR9 for thesurvival of mice is less clear. TLR9-deficient mice showed either nodifference in survival in infection models using HSV or, limitedsurvival differences within a narrow range, in infection models usingmouse cytomegalovirus (MCMV) (Krug et al., Blood 103:1433-1437 (2004);Tabeta et al., Proc. Natl. Acad. Sci. U.S.A. 101:3516-3521 (2004);Delale et al., J. Immunol. 175:6723-6732 (2005)). Given the strongsuppression of TLR9-independent recognition in vitro by poxviruses likeECTV (FIG. 1, FIG. 2), it was hypothesized that TLR9 would be animportant factor for the survival of infection with these viruses. Totest this, a mouse infection model that mimicked as closely as possiblea human smallpox infection was used: an ECTV infection model via theintranasal route. Similar to VARV infection in humans, ECTV is highlyspecies specific and is a natural mouse pathogen, able to effectivelyinfect via the respiratory tract after exposure with only small viraldoses. In addition, it carries a large panel of immune suppressivemolecules similar to VARV (Esteban, D. J., and Buller, R. M., J. Gen.Virol. 86:2645-2659 (2005)).

Initial experiments using relatively high doses of ECTV (1E+04 tissueculture infective doses (TCID₅₀)) demonstrated that TLR9-deficient micedied at least 2 days earlier than wild type mice. To further evaluatethe susceptibility and quantify the _(LD50) TLR9-deficient and wild typemice were infected with varying doses of ECTV. All TLR9-deficient micedied after infection with as little as 3E+01 TCID₅₀ whereas none diedafter inoculation with 1E+01 TCID₅₀ (FIG. 3 b). In contrast, none of thewild type mice died after infection with 1E+02 TCID₅₀ and only whenusing 1E+04 TCID₅₀ all mice succumbed to Ectromelia infection (FIG. 3a). There was some variation between experiments with wild type miceusing the doses of 3E+02 to 3E+03 TCID₅₀, which was partiallygender-specific, with male mice being more susceptible than female mice.An _(LD50) of 19 TCID₅₀ for the TLR9-deficient mice and an _(LD50) ofabout 2120 TCID₅₀ for the wild type mice was calculated. Thus,TLR9-deficient mice are more than 100-fold more susceptible to ECTVinfection than wild type mice. Therefore, in strong agreement with thein vitro data, TLR9 is an essential component of the immune response toECTV infection.

Example 6 MVA Immediately Protects Wild Type and TLR9-Deficient Micefrom Lethal ECTV Challenge

In vitro experiments demonstrated that ECTV effectively suppressedrecognition by DC, whereas MVA activated DC (FIG. 1). It was thereforehypothesized that MVA given at the same time as the pathogen wouldactivate the immune system and as a result might induce immune responseswhich potentially control the pathogenic poxvirus. Indeed, MVA given atthe same time or immediately after challenge with a high lethal dose ofECTV of 1E+05 TCID₅₀ completely protected wild type mice against deathwhereas all control mice died with the 10-fold lower dose of 1E+04TCID₅₀ (FIG. 4).

Since MVA induced a strong TLR9-independent activation of immune cellsin vitro, whether MVA could protect TLR9-deficient mice against ECTVinfection was tested next. Similar to the protection seen in wild typemice (FIG. 4), MVA immediately protected TLR9-deficient mice againsthighly lethal doses of ECTV infection. Whereas all untreated controlmice died with 1E+02 TCID₅₀, all MVA treated mice even survived achallenge with 1E+04 TCID₅₀, which resembles a dose exceeding 500-foldthe _(LD50) for TLR9-deficient mice (FIG. 5). It was observed thatTLR9-deficient mice challenged with high doses of ECTV (3E+03 and 1E+04TCID₅₀) developed tail lesions after 2-3 weeks which disappeared after 4weeks. The tail lesions on otherwise symptom free TLR9-deficient miceindicated that MVA-induced immune responses were able to prevent severeECTV-induced disease and death, but not to completely eliminate thevirus within the first weeks.

Example 7 Mice can be Protected Against Lethal ECTV Infection if MVA isApplied to a Different Site

To ascertain whether the immediate protection in wild type andTLR9-deficient mice was absolutely dependent on the coadministration ofMVA to the same site as the ECTV infection mice were challengedintranasally with a lethal dose of ECTV and applied MVA via asubcutaneous injection. The TLR9-deficient (FIG. 6 a) and wild type mice(FIG. 6 b) survived the lethal ECTV infection, without any signs ofsickness, if MVA was applied to the subcutaneous site (FIG. 6). Thuscoadministration of MVA to the same site as ECTV was not essential forimmediate protection.

Example 8 Inactivated Orthopoxviruses are Less Efficient than MVA inProtection from Lethal ECTV Infection

Our in vitro experiments have suggested that UV-inactivatedorthopoxviruses act as exclusive TLR9 agonists but have lost theirability to stimulate via a TLR9-independent way (FIG. 2). To test ifthis ‘TLR-9 only’ stimulation in the presence of orthopoxvirus-antigenwould mount any protection wild type mice were challenged with a lethaldose of ECTV and applied subcutaneously the equivalent of 1E+08 TCID₅₀of a UV-inactivated CVA. Of the 5 mice challenged one died on day 11whereas the others survived (FIG. 6 b). However in contrast to the mirewhich received the same dose of active MVA subcutaneously, all micetreated with inactivated CVA showed strong signs of sickness includinglethargy and they developed tail lesions which healed only in the 4^(th)week of challenge. Thus inactivated orthopoxviruses, although providingviral antigen and potential TLR9 ligand, seem to induce protection thatis inferior to the robust protection achieved with active MVA.

Example 9 MVA Mediated Immediate Protection from Lethal ECTV Challengeis Partially Independent of IFN-I

To elucidate if administration of MVA was able to immediately protectother immune compromised mice and to shed light on the mechanism ofprotection experiments were performed with IFN-I receptor(IFN-I-R)-deficient mice, which are known to be highly susceptible toseveral viral infections including poxvirus infections (26). Initialexperiments demonstrated that similar to TLR9-deficient mice allIFN-I-R-deficient mice died after challenge with 1E+02 TCID₅₀ of ECTV.Since the IFN-α production in vitro to ECTV but not to MVA was dependenton TLR9, it was hypothesized that MVA induced IFN-α was an essentialpart of the immediate protection in TLR9-deficient mice. However,whereas the untreated control IFN-I-R-deficient mice died with achallenge of 1E+02 TCID₅₀ ECTV within 10 days, immediate MVA treatmentsurprisingly protected the IFN-I-R-deficient mice against a challengewith 1E+02 and 1E+03 TCID₅₀ ECTV (FIG. 7). From a total of 15 IFN-I-Rmice challenged with 1E+02 TCID₅₀ of ECTV, one mouse developed a swollenlimb and had to be euthanized after 3 weeks for ethical reasons, whereasthe other 14 mice and all 10 mice challenged with 1E+03 ECTV were freeof symptoms for more than 4 weeks. However with higher doses of ECTV theprotection of IFN-IR-deficient mica was much less robust. About half ofthe IFN-I-R mice challenged with 1E+04 ECTV died and all IFN-I-R micechallenged with 1E+05 ECTV died (FIG. 7). Since these higher dosescorrespond to viral challenges that wild type mice on the samebackground could survive in the presence of MVA, it was concluded thatone mechanism of the immediate protection via MVA is mediated by IFN-I.However, some protection is clearly mediated via IFN-I-independentmechanisms since MVA could protect mice against low and intermediatedoses of lethal ECTV infection even in the absence of a functional IFN-Isystem.

Example 10 The Immediate Protection Via MVA in ECTV Infection Depends onAdaptive Immune Responses

MVA is known to induce strong adaptive immune responses includingcytotoxic T-cell (CTL) responses and antibody formation which bothcontribute to the protection against pathogenic orthopoxviruses (Wyattet al., Proc. Natl. Acad. Sci. U.S.A. 101:4590-4595 (2004)). Previously,it has been shown that TLR9-deficient mice are able to mount stable CTLand antibody responses upon DNA vaccination, thus demonstrating theoverall capability of these mice to mount solid adaptive immuneresponses (Spies et al., J. Immunol. 171:5908-5912 (2003); Babiuk etal., Immunology 113:114-120 (2004)).

To test if the absence of TLR9 would affect adaptive immune responses topoxviruses MVA was applied and antibodies to poxviruses were measured byELISA in the serum and poxvirus specific CTL responses by pentamerstaining to B8R in spleen cells and peripheral blood cells.TLR9-deficient mice mounted robust poxvirus specific antibody and CTLresponses indicating that adaptive immune responses in response to MVAvaccination are not dependent on the presence of TLR9.

It was next investigated if the measured adaptive immune responses wouldtranslate into long lasting protection to ECTV infection, thus whetherthe MVA-induced protection in TLR9-deficient mice was not only immediate(FIG. 5), but also long-lasting. Nine weeks after initial challenge, theTLR9-deficient mice from the experiments described above (FIG. 5) and inaddition mice that had received MVA alone nine weeks earlier werere-challenged using 1E+04 TCID₅₀ of ECTV. All TLR9-deficient mice whichhad received a single dose of MVA nine weeks earlier either alone or incombination with ECTV survived the challenge with 1E+04 TCID₅₀ of ECTV.As observed with the immediate protection, the long lasting protectionof the TLR9-deficient mice after MVA treatment exceeded a factor of 500of the _(LD50). These experiments demonstrated that TLR9-deficient micewere capable of mounting and sustaining substantial protective immunityto poxvirus infection upon traditional vaccination with MVA which mostlikely depended on adaptive immune responses.

To prove the role of adaptive immune responses in the immediateprotection protocol Rag-1 deficient mice were challenged with ECTV inthe presence or absence of MVA (FIG. 8). Rag-1 deficient mice lackmature B-cells and T-cells and thus are unable to produce antibodies andCTL. Without co administration of MVA Rag-1 deficient mice died rapidlyin response to ECTV challenge (1E+02 and 1E+03). Cotreatment with MVAextended the survival of Rag-1 mice for several days, but finally allmice died, demonstrating that adaptive immune responses are indeedcrucial for the survival of ECTV, even in the presence of immediatelyapplied MVA.

Example 11 MVA Fully Rescues TLR9-Deficient Mice if applied Two DaysAfter Infection with ECTV

The WHO recommendation in cases of smallpox infection includesvaccination as quickly as possible after exposure. However there existsonly anecdotal historical information about the success of post-exposurevaccination against smallpox and in most cases the pre-vaccinationstatus of the individuals was not clear. (Fenner, F., Henderson, D. A.,Arita, I., Jezek, Z., & Ladnyi, I. D. Smallpox and its eradication.(Geneva: World Health Organization; 1988); Mortimer, P. P., Clin.Infect. Dis. 36, 622-629 (2003)). Moreover, in animal models nosignificant survival benefit to post-exposure vaccination was observedusing as infection models either the MPXV in monkeys or VACV in mice(Stittelaar et al., Nature 439:745-748 (2006); Staib et al., J. Gen.Virol. 87:2917-2921 (2006)). In this way, the results of the currentinvention are unexpected.

Given this scenario and the fact that the intranasal infection model ofECTV is regarded as a good animal model for smallpox infection in humans(Esteban, D. J., and Buller, R. M., J. Gen. Virol. 86:2645-2659 (2005)),whether the robust immediate protection against a lethal ECTV infectionby MVA could be extended to a therapeutic post-exposure interventionwith MVA was analyzed. As shown in FIG. 9, MVA given up to two daysafter exposure to a lethal dose of ECTV, completely protectedTLR9-deficient mice against death without any obvious signs of sickness(FIG. 9 and data not shown). Some mice in the group which received MVAtreatment as late as 3 days after a lethal dose of ECTV also survived(FIG. 9 b). These data show protection against death to species-specificorthopoxvirus infection using as a post-exposure treatment.

1-40. (canceled)
 41. A method for inducing an immune response against aninfectious agent in an animal, comprising administering to the animal animmunogenic composition comprising a poxvirus between 36 hours prior toinfection with the infectious agent and 72 hours after infection withthe infectious agent, wherein said poxvirus is replication incompetentin the animal.
 42. The method of claim 41, wherein the immunogeniccomposition comprising a poxvirus is administered between 36 hours priorto infection with the infectious agent and 48 hours after infection withthe infectious agent.
 43. The method of claim 41, wherein the animal isa human.
 44. The method of claim 41, wherein the poxvirus is a ModifiedVaccinia Virus Ankara (MVA).
 45. The method of claim 41, wherein theinfectious agent is a replication competent poxvirus.
 46. The method ofclaim 44, wherein the MVA is administered in a dose of 10⁵ to 5×10⁸TCID50.
 47. The method of claim 46, wherein the MVA is administered in adose of 10⁷ to 5×10⁸ TCID50.
 48. The method claim 44, wherein the MVA isadministered intravenously, intranasally, intramuscularly, orsubcutaneously.
 49. The method of claim 44, wherein the MVA is MVA-BN®.50. The method of claim 44, wherein the MVA is a recombinant MVA. 51.The method of claim 50, wherein the MVA comprises at least oneheterologous nucleic acid sequence coding for at least one antigenicepitope.
 52. The method of claim 51, wherein the antigenic epitope is anantigenic epitope of the infectious agent.
 53. The method of claim 52,wherein the infectious agent is selected from viruses, fungi, pathogenicunicellular eukaryotic and prokaryotic organisms, and parasiticorganisms.
 54. The method claim 53, wherein the virus is selected fromInfluenza virus, Flavivirus, Paramyxovirus, Hepatitis virus, humanimmunodeficiency virus, and viruses causing hemorrhagic fever.
 55. Themethod of claim 53, wherein the infectious agent is bacillus anthracis.56. The method of claim 42, wherein the immunogenic composition isadministered between 24 hours prior to infection with the infectiousagent and 48 hours after infection with the infectious agent.
 57. Themethod of claim 56, wherein the immunogenic composition is administeredbetween 0 and 24 hours prior to infection with an infectious agent. 58.The method of claim 56, wherein the immunogenic composition isadministered between 0 and 48 hours after infection with an infectiousagent.
 59. A kit for inducing an immune response against an infectiousagent in an animal comprising: (a) an immunogenic composition comprisinga poxvirus, wherein said poxvirus is replication incompetent in saidanimal; and (b) instructions that said immunogenic composition is to bedelivered to said animal at a time point between 0 hours and 72 hoursafter exposure to an infections agent.
 60. The kit of claim 59, whereinsaid immunogenic composition is to be delivered to said animal,including a human, at a time point between 0 hours and 36 hours prior toexposure to an infectious agent.
 61. The kit of claim 59, wherein saidimmunogenic composition is to be delivered to said animal, including ahuman, at a time point between 0 hours and 72 hours after exposure to aninfectious agent.
 62. The kit of claim 59, wherein said poxvirus is aModified Vaccinia Virus Ankara (MVA).
 63. The kit of claim 62, whereinthe MVA is MVA-BN®.
 64. The kit of claim 63, wherein the MVA is MVA-BN®at a dose of 10⁵ to 5×10⁸ TCID50.
 65. The kit of claim 62, wherein theMVA is a recombinant MVA.
 66. The kit of claim 59, wherein theinfectious agent is smallpox.
 67. The kit of claim 59, wherein theinfectious agent is bacillus anthracis.
 68. A kit comprising animmunogenic composition comprising an MVA and instructions to deliverthe immunogenic composition to a human as soon as possible afterexposure to smallpox.